Therapeutic proteins are the treatment modality of choice for many disorders. Therapeutic proteins have a more clearly defined action mechanism than small molecule drugs. Furthermore, they can be administered using methods that are safer than methods for directly increasing protein expression, such as gene therapy. Advances in biotechnology have accelerated the economical, large-scale production of proteins, vaccines, and hormones, making them readily available for therapeutic applications in medical practices and clinical studies. In recent years, the market penetration of the pharmaceutical protein industry gained substantial momentum. To date over 140 FDA-approved protein drugs have been placed on the market, and more are coming. It would be desirable to be able to deliver such therapeutic proteins and other therapeutic agents through non-invasive oral or transmucosal routes.
As an example, phenylketonuria (PKU), the most common inborn metabolic disorder in the world, affecting 1 of every 1,000 people, is a genetic disorder caused by a mutation in the gene that encodes for the enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the amino acid phenylalanine (Phe) to the amino acid tyrosine (Tyr). When a mutation renders this vital enzyme nonfunctional, Phe, which is present in almost every food containing protein, cannot be processed. Phe builds to dangerously high concentrations in the brain and bloodstream in those afflicted with this disease. Left untreated, it causes a wide range of acute cognitive and behavioral defects. These include severe mental retardation, aggression, and seizures.
Delivering phenylalanine ammonia lyase (PAL), a plant enzyme that converts phenylalanine to non-toxic metabolites ammonia and trans-cinnamic acid, is a potential therapy to compensate for the lack of intrinsic PAH. As PKU is a digestion related disease, orally delivering PAL may present an effective noninvasive treatment for PKU that does not require penetration through the gastrointestinal epithelial membrane.
Another example is the possible oral delivery of alcohol degrading enzyme to relieve the pain resulting from alcohol abuse. Alcohol abuse is associated with a variety of organ injuries (such as liver damage), as well as with serious social problems (such as violence and driving under the influence of alcohol (DUI)). Providing a way to quickly eliminate alcohol in-vivo would be a significant step towards solving these problems.
Ethanol-degrading enzymes, which have a well-defined detoxification mechanism and low toxicity, are the most direct and safe antidotes to alcohol abuse. However, the effectiveness of alcohol dehydrogenase is limited by the low extracellular concentration of NAD+. Thus it cannot serve as an effective antidote when the alcohol concentration is high. Furthermore, the associated consumption of NAD+ may cause many undesired metabolic regulations due to alterations of redox status inside a cell, affecting various biological pathways.
In contrast, alcohol oxidase (AOx), which employs molecular oxygen as a substrate, is capable of achieving rapid clearance of alcohol. Thus, delivering AOx orally would be an effective and non-invasive way to detoxify alcohol.
In addition to PAL and AOx, a number of other therapeutic proteins have shown great promise in clinical applications. Together with other protein drugs, such as insulin, calcitonin, interferons, human growth hormone, glucagons, gonadotropin-releasing hormones, encephalin, vaccines, enzymes, hormone analogs, and enzyme inhibitors, these protein therapeutics have a vast market worldwide.
Despite its fast growth in recent years, protein therapy is still in its adolescence. There are a number of reasons for this, including the difficulties associated with delivering functional proteins that retain their catalytic activity, poor protein stability both in vitro and in vivo, and the high degradation and clearance rates associated with protein therapy. These problems are especially significant in the oral administration of therapeutic proteins. Oral administration of drugs is favorable over other routes of administration due to its simplicity and convenience. However, administrating therapeutic proteins orally is problematic, due to the harsh acidic environment in the stomach, the existence of various proteases in relatively high concentrations, and poor oral adsorption of proteins. Strategies to address these issues are ongoing and in high demand in the biotechnology and pharmaceutical industries.
Various strategies have been attempted to improve the oral bioavailability of therapeutic proteins. The main approaches presently include 1) using absorption enhancers to improve the lipophilicity of the protein to favor transmucosal delivery, 2) using protease inhibitors to tune down enzymatic digestion of the protein, and 3) utilizing mucoadhesive polymeric systems to increase the resident time of the protein in GI tract. To date, various oral delivery systems for proteins have been actively developed, especially by pharmaceutical companies in the hopes of rendering various therapeutic proteins clinically useful. Although these attempts have in some cases achieved enhanced protein stability and/or improved transmucosal protein delivery, none of them have been proven to be good enough for clinical applications.
In the case of PKU treatment, investigators in the United Kingdom found that administrating PAL in enteric-coated capsules could reduce blood phenylalanine levels by 25%. Additionally, a Canadian group has been trying to encapsulate PAL in semipermeable microcapsules and to deliver the resulting microcapsules enterically. Ideally, the enterically placed PAL would metabolize dietary phenylalanine before it is absorbed, and phenylalanine in the blood could recirculate into the intestinal lumen to be degraded by the PAL. The enteric delivery of these microcapsules achieved a 50% reduction in the blood phenyalanine concentration in PAHenu2 mice. Although the degree of reduction achieved by this method is insufficient for the adequate treatment of PKU, these results validated the safety and therapeutic potential of enterically delivered PAL.
Further, although alcohol oxidase (AOx) has great potential as an alcohol detoxification agent, it is not well studied in the context of pharmaceutical applications. In the 1980s, it was shown that by injecting alcohol oxidase post alcohol intoxication, blood alcohol concentration could be reduced by up to three fold. However, the large dosage required, high cost, and resulting immune response blocked the further development of AOx-related drugs. As a consequence, little work has been done on the development of AOx-based alcohol detoxification agents, including the oral delivery of AOx.
Accordingly, there is a need in the art for methods and compositions that can be used to effectively overcome the problems associated with the oral delivery of therapeutic agents, such as, functional therapeutic proteins. Specifically, such compositions and methods should enable retention of catalytic activity, protect the therapeutic agent from acid attack and protease degradation, facilitate the absorption of the therapeutic agent, and be non-toxic and biocompatible.